Bull Vet Inst Pulawy 48, 201-205, 2004 PRION PROTEIN GLYCOFORMS FROM BSE CASES IN POLAND MIROSŁAW P. POLAK, WOJCIECH ROŻEK, JERZY ROLA AND JAN F. ŻMUDZIŃSKI Department of Virology, National Veterinary Research Institute, 24-100 Pulawy, Poland e-mail: ppolak@piwet.pulawy.pl Received for publication May 12, 2004. Abstract The article describes the analysis of proteinase K resistant prion protein from Polish cases of bovine spongiform encephalopathy (BSE). Brainstem samples from 12 out of 14 cases of BSE diagnosed so far in Poland have been used in the study. Glycotyping showed comparable molecular sizes of three glycoforms in all cases and typical for BSE predominance of diglycosylated form of PrP res in 11 samples. In one case equivalent relative amounts of both di- and monoglycosylated forms of prion protein were detected. The results were compared with both human and other animal TSEs glycotyping studies of PrP res and the reference was made to the possibility of existence of BSE strains on the basis of characteristic glycoforms of PrP res . Key words: bovine spongiform encephalo- pathy, glycoforms, PrP res typing, prion strains. Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle with characteristic lack of immunological response, extended incubation period and 100% fatal outcome. Scrapie in sheep and Creutzfeldt-Jakob disease in humans are other diseases belonging to the same group of neurodegenerative disorders, also known as prion diseases (13). The hallmark of BSE pathogenesis is the post- translational conversion of host-encoded prion protein (PrP C ) to its pathological isomer defined as PrP Sc or PrP res according to its partial resistance to proteolysis. The prion protein has two N glycosylation sites and is present in the cell in three glycoforms (di-, mono-, and unglycosylated form) (8). In human TSEs it has been shown that differences in PrP res glycosylation may be responsible for strain-specific features of prion protein defining specific biological and biochemical properties (5, 7, 12). Histopathology, immunohistochemistry and western-blot can be used to characterize localisation and magnitude of spongiform changes, length of incubation period in experimental rodent models, and PrP res glycosylation pattern (relative amount of di-, mono-, and unglycosylated forms of PrP res as well as their sizes defined by migration distance in SDS-PAGE gel). Until recently, all available tests used to characterise BSE causative agent showed the same histopathological lesion profile, the same incubation period and similar glycotypes which indicated the existence of a single strain of BSE (1, 9, 11). Western blot analysis of PrP res glycoforms from BSE cases showed that the diglycosylated fragment was the most abundant and the size of the unglycosylated form defined by its electrophoretic mobility was unchanged. The opposite was for scrapie and CJD where, at least several different strains were described by means of the same criteria (2- 5, 7, 10, 12, 15). In March 2004, a group of Italian researchers described a molecular variant of bovine spongiform encephalopathy (6). Opposite to typical BSE, monoglycosylated form was predominant and all forms of PrP res showed faster electrophoretic mobility. These features were similar to PrP res of sporadic Creutzfeldt- Jakob disease. This discovery indicated the possibility of existence of a new strain of BSE potentially pathogenic for humans (probable appearance of a new disease in relation to BSE, like variant CJD). Until the end of April 2004, 14 cases of BSE have been detected in Poland, all in native born cattle. Approximately 1 million samples have been tested so far within active monitoring framework using rapid tests. While 11 from 14 BSE cases discovered in Poland came from healthy animals, slaughtered for human consumption, only three cases detected so far were diagnosed in risk animals. Two of these cases were found in clinical suspects and one was diagnosed in dead farm animal. The objective of the study was to compare Polish cases of BSE at molecular level in terms of electrophoretic rate of migration and glycoform content of prion protein in respect to the possible existence of molecular variants of pathological form of PrP resistant to proteinase K digestion. Two cases (the 4 th and 5 th case) were not analysed (brainstem samples were formalin-fixed).